Heterodimeric proteins and methods for producing and purifying them

A cell line expressing a heterodimeric protein, wherein the heterodimeric protein comprises:

• a hinge region comprising a first immunoglobulin-like hinge polypeptide and a second immunoglobulin-like hinge polypeptide which interact together to form a dimeric hinge interface, wherein electrostatic interactions between one or more charged amino acids within the hinge interface favor interaction between the first and second hinge polypeptides over interaction between two first hinge polypeptides or two second hinge polypeptides, thereby promoting heterodimer formation over homodimer formation, wherein the hinge region is a human IgG1 or IgG2 hinge region, wherein the first hinge polypeptide comprises at least one amino acid modification relative to a wild-type IgG hinge region;
• wherein the wild-type amino acid in the first hinge polypeptide is replaced with an amino acid having an opposite charge to the corresponding amino acid in the second hinge polypeptide, wherein the amino acid modification in the hinge region is at a position selected from the group consisting of 1) Asp221 or 2) Cys223 and/or Glu225 as shown at FIG. 6A, and further comprising an immunoglobulin-like CH3 region comprising a first CH3 polypeptide fused to the first hinge polypeptide and a second CH3 polypeptide fused to the second hinge polypeptide, wherein the first CH3 polypeptide and the second CH3 polypeptide comprise at least one amino acid modification relative to a wild-type IgG1 CH3 region sequence at a position selected from the group consisting of Tyr349, Leu368, Phe405, and Lys409 (EU numbering scheme) as shown at FIG. 10.